The ability to create functional vasculatures is a crucial step towards vascular therapy and tissue engineering. Currently, we still do not have full control and understanding of vascular differentiation including endothelial cells (ECs) and pericytes, from human induced pluripotent stem cells (hiPSCs). During embryogenesis, vasculogenesis gives rise to the primitive plexus followed by angiogenesis where ECs sprout, elongate, lumenize and coalesce to form tubes. Concurrently, these nascent ECs tubes are stabilized via the recruitment of perivascular cells. Traditionally, engineering of functional microvasculature constructs in vitro involves embedding two distinct cell sources of ECs and pericytes within a three-dimensional (3D) scaffold material. Our lab has established a novel adherent culture system to derive a bi-cellular population of early vascular cells (EVCs) from hiPSCs. These EVCs are composed of VEcad+ and PDGFR cells that can mature to ECs and pericytes respectively, and can self-organize to a 3D multicellular vascular network in a hydrogel scaffold. This approach provides great opportunities to study fate decisions during vascular differentiation of hiPSCs. It also contributes to the construction of vascular structures for clinical applications. Our aims are: (1) Establish a feeder free, adherent culture system to obtain high ratio of EC to pericyte from hiPSCs; (2) Dissect the spatial and temporal kinetics of EC and pericyte differentiation; and (3) Analyze real- time cellular interactions and functionality of self-organized vascular networks. To achieve our aims, the proposed research strategy combines techniques in stem cell and vascular biology engineering. Successful completion of these aims has considerable clinical impact with respect to improved vascular therapeutics and will broaden our understanding of vascular development and repair.